Nucleic acid molecules such as microRNAs (miRNAs) are targets for detection in various medical and scientific applications.
MiRNAs are a class of short (18-24 nucleotides) non-coding RNA molecules that negatively regulate gene expression at the post transcriptional level (Ambros, V. (2004). Nature, 431(7006), 350-355; Bartel, D. P. (2004). Cell, 116(2), 281-297). They play important roles in a variety of physiological and pathological processes, such as development, differentiation, cell proliferation, apoptosis, and stress responses (Kloosterman, W. P., & Plasterk; R. H. (2006). Dev Cell, 11(4), 441-450; Stefani, G., & Slack, F. J. (2008). Nat Rev Mol Cell Biol, 9, 219-230). Though miRNAs have originally emerged as cell-based biomarkers, recent reports indicated that they exist in cell-free form as well (Chim, S. S. C., et al. (2008). Clin Chem, 54, 482-490; Gilad, S., et al. (2008). PLoS One, 3(9), e3148-e3154). In fact, extra-cellular miRNAs have been detected in multiple body fluids including plasma, serum, saliva, and urine (Hanke, M., et al. (2010). Urol Oncol, 28, 655-661; Weber, J. A., et al. (2010). Clin Chem, 56(11), 1733-1741). Aberrant expression levels of such circulating miRNAs have been associated with cancer (Calin, G. A., & Croce, C. M. (2006). Nat Rev Cancer, 6, 857-866; Esquela-Kerscher, A., & Slack, F. J. (2006). Nat Rev Cancer, 6(4), 259-269; Zhao, H., et al. (2010). PLoS One, 5(10), e13735-e13746), cardiovascular disorders (Corsten, M. F., et al. (2010). Circ Cardiovasc Genet, 3(6), 499-506), abnormal pregnancies (Zhang, Y., et al. (2009). Am J Obstet Gynecol, 202, e1-7), diabetes (Zampetaki, A., et al. (2010). Circ Res, 107(6), 810-817), autoimmune diseases (Wang, G., et al. (2010). J Rheumatol, 37(12), 2516-2522), and so on. Given the facts that circulating miRNAs are highly stable (Chen, X., et al. (2008). Cell Res, 18(10), 997-1006), e.g. resistant to RNase digestion, and that their expression levels are reproducibly consistent among individuals of the same species, they show promises as novel and non-invasive biomarkers for disease detection.
With the emergence of miRNAs as key players in disease diagnosis and screening, the development of rapid, sensitive, and quantitative miRNA detection is of high interest. Chip-based devices have been used for the ultra-trace detection of analytes, and the development of a nucleic acid-based microarray is particularly useful for high-throughput detection and profiling of miRNA targets. However, the small size of miRNAs poses a major challenge toward developing a reliable assay. For a short target-probe hybrid, the oligonucleotide annealing temperature is low, thereby lessening the stringency of hybridization and greatly increasing the risk of false-positive signals. Selective detection of miRNA is further challenging due to high sequence homology among family members, which is as close as a single base variation. In addition, many existing technologies for RNA detection necessitate extensive sample preparation (e.g. RNA isolation, PCR amplification, tagging etc.).
Therefore, there is still need in the art for improved methods that overcome the drawbacks of existing techniques.